1 RT ± PCR technique was used to clone the human 5-HT 4(e) receptor (h5-HT 4(e) ) from heart atrium. We showed that this h5-HT 4(e) receptor splice variant is restricted to brain and heart atrium. 2 Recombinant h5-HT 4(e) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg 71 protein, respectively. Expression of h5-HT 4(e) receptors at the cell membrane was con®rmed by immunoblotting. 3 The receptor binding pro®le, determined by competition with [3 H]-GR113808 of a number of 5-HT 4 ligands, was consistent with that previously reported for other 5-HT 4 receptor isoforms. Surprisingly, we found that the rank order of potencies (EC 50 ) of 5-HT 4 agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of anities (K i ) obtained from binding assays. Furthermore, EC 50 values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. 4 ML10302 and renzapride behaved like partial agonists on the h5-HT 4(e) receptor. These results are in agreement with the reported low ecacy of the these two compounds on L-type Ca 2+ currents and myocyte contractility in human atrium. 5 A constitutive activity of the h5-HT 4(e) receptor was observed in CHO cells in the absence of any 5-HT 4 ligand and two 5-HT 4 antagonists, GR113808 and ML10375, behaved as inverse agonists. 6 These data show that the h5-HT 4(e) receptor has a pharmacological pro®le which is close to the native h5-HT 4 receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed.

G-protein-coupled receptor dimerization directs the design of new drugs that specifically bind to receptor dimers. Here, we generated a targeted series of homobivalent ligands for serotonin 5-HT(4) receptor (5-HT(4)R) dimers composed of two 5-HT(4)R-specific ML10302 units linked by a spacer. The design of spacers was assisted by molecular modeling using our previously described 5-HT(4)R dimer model. Their syntheses were based on Sonogashira-Linstrumelle coupling methods. All compounds retained high-affinity binding to 5-HT(4)R but lost the agonistic character of the monomeric ML10302 compound. Direct evidence for the functional interaction of both pharmacophores of bivalent ligands with the 5-HT(4)R was obtained using a bioluminescence resonance energy transfer (BRET) based assay that monitors conformational changes within 5-HT(4) dimers. Whereas the monovalent ML10302 was inactive in this assay, several bivalent derivatives dose-dependently increased the BRET signal, indicating that both pharmacophores functionally interact with the 5-HT(4) dimer. These bivalent ligands may serve as a new basis for the synthesis of potential drugs for 5-HT(4)-associated disorders.